thioacetamide and Cell-Transformation--Neoplastic

Cholangiocarcinoma (CCA) is the second most common primary liver malignancy and carries a dismal prognosis due to difficulties in achieving an optimal resection, and poor response to current standard-of-care systemic therapies. We previously devised a CTLA4-PD-L1 DNA cancer vaccine (DNA vaccine) and demonstrated its therapeutic effects on reducing tumor growth in a thioacetamide (TAA)-induced rat intrahepatic CCA (iCCA) model. Here, we developed a CTLA4-PD-L1 chimeric protein vaccine (Protein vaccine), and examined its effects in the rat iCCA model. In a therapeutic setting, iCCA-bearing rats received either DNA plus Protein vaccines or Protein vaccine alone, resulting in increased PD-L1 and CTLA-4 antibody titers, and reduced iCCA tumor burden as verified by animal positron emission tomography (PET) scans. Treating iCCA-bearing rats with Protein vaccine alone led to the increase of CTAL4 antibody titers that correlated with the decrease of tumor SUV ratio, indicating regressed tumor burden, along with increased <i>CD8</i> and granzyme A (<i>GZMA</i>) expression, and decreased PD-L1 expression on tumor cells. In a preventive setting, DNA or Protein vaccines were injected in rats before the induction of iCCA by TAA. Protein vaccines induced a more sustained PD-L1 and CTLA-4 antibody titers compared with DNA vaccines, and was more potent in preventing iCCA tumorigenesis. Correspondingly, Protein vaccines, but not DNA vaccines, downregulated PD-L1 gene expression and hindered the carcinogenesis of iCCA. Taken together, the CTLA4-PD-L1 chimeric protein vaccine may function both as a therapeutic cancer vaccine and as a preventive cancer vaccine in the TAA-induced iCCA rat model.

Topics: Animals; B7-H1 Antigen; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cancer Vaccines; Carcinogenesis; Cell Transformation, Neoplastic; Cholangiocarcinoma; CTLA-4 Antigen; Immune Checkpoint Proteins; Rats; Recombinant Fusion Proteins; Thioacetamide

Most hepatocellular carcinomas (HCCs) develop in the context of chronic liver inflammation. Oxidative stress is thought to play a major role in the pathogenesis of HCC development. In this study, we examined whether cold-inducible RNA-binding protein (Cirp) controls reactive oxygen species (ROS) accumulation and development of HCC by using murine models of hepatocarcinogenesis and human liver samples. Cirp expression, ROS accumulation, and CD133 expression were increased in the liver of tumor-harboring mice. Cirp deficiency reduced production of interleukin-1β and interleukin-6 in Kupffer cells, ROS accumulation, and CD133 expression, leading to attenuated hepatocarcinogenesis. Thioacetamide treatment enhanced hepatic expression of CD133 and phosphorylated signal transducer and activator of transcription 3 (STAT3), which was prevented by treatment with the antioxidant butylated hydroxyanisole. Intriguingly, the risk of human HCC recurrence is positively correlated with Cirp expression in liver. Cirp appears to play a critical carcinogenic function and its expression might be a useful biomarker for HCC risk prediction.

Topics: AC133 Antigen; Animals; Antigens, CD; Antioxidants; Butylated Hydroxyanisole; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Glycoproteins; Humans; Interleukin-1beta; Interleukin-6; Kupffer Cells; Liver; Liver Neoplasms; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Recurrence, Local; Neoplastic Stem Cells; Oxidative Stress; Peptides; Phosphorylation; Reactive Oxygen Species; RNA-Binding Proteins; SOXB1 Transcription Factors; STAT3 Transcription Factor; Thioacetamide; Tissue Fixation

Chronic inflammation triggers the aberrant expression of a DNA mutator enzyme, activation-induced cytidine deaminase (AID), and contributes to tumorigenesis through the accumulation of genetic aberrations. To gain further insight into the inflammation-mediated genotoxic events required for carcinogenesis, we examined the role of chronic inflammation in the emergence of genetic aberrations in the liver with constitutive AID expression. Treatment with thioacetamide (TAA) at low-dose concentrations caused minimal hepatic inflammation in both wild-type (WT) and AID transgenic (Tg) mice. None of the WT mice with low-dose TAA administration or AID Tg mice without hepatic inflammation developed cancers in their liver tissues over the 6 month study period. In contrast, all the AID Tg mice with TAA treatment developed multiple macroscopic hepatocellular carcinomas during the same observation period. Whole exome sequencing and additional deep-sequencing analyses revealed the enhanced accumulation of somatic mutations in various genes, including dual specificity phosphatase 6 (Dusp6), early growth response 1 (Egr1) and inhibitor of DNA binding 2 (Id2), which are putative tumor suppressors, in AID-expressing liver with TAA-mediated hepatic inflammation. Microarray and quantitative reverse transcription-polymerase chain reaction analyses showed the transcriptional upregulation of various genes including Dusp6, Egr1 and Id2 under hepatic inflammatory conditions. Together, these findings suggest that inflammation-mediated transcriptional upregulation of target genes, including putative tumor suppressor genes, enhances the opportunity for inflamed cells to acquire somatic mutations and contributes to the acceleration of tumorigenesis in the inflamed liver tissues.

Topics: Animals; Cell Transformation, Neoplastic; Cytidine Deaminase; Dose-Response Relationship, Drug; Dual Specificity Phosphatase 6; Early Growth Response Protein 1; Gene Expression Regulation; Hepatitis, Chronic; Inhibitor of Differentiation Protein 2; Liver Neoplasms; Mice, Inbred C57BL; Mice, Transgenic; Mutagenesis; Mutation; Thioacetamide

Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1(-/-) mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Chemokine CCL2; Cytokines; Diethylnitrosamine; Hepatocytes; Humans; Inflammation; Lectins, C-Type; Lipopolysaccharide Receptors; Lipopolysaccharides; Liver; Liver Cirrhosis; Liver Neoplasms; Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred C57BL; Mice, Knockout; Recombinant Proteins; Sepsis; Signal Transduction; Thioacetamide; Toll-Like Receptor 4; Up-Regulation

Thioacetamide (TAA) induces oxidative stress and hepatocarcinogenicity in rats. We previously reported that TAA promotion caused various disruptions in cell cycle protein expression in rats, including downregulation of p16(Ink4a), which is associated with intraexonic hypermethylation in hepatocellular proliferative lesions. This study further investigated the contribution of cell cycle aberrations associated with early hepatocarcinogenic processes induced by TAA using antioxidants, enzymatically modified isoquercitrin (EMIQ) and α-lipoic acid (ALA), in a two-stage rat hepatocarcinogenesis model. TAA-promotion after initiation with N-diethylnitrosamine increased the number and area of hepatocellular foci immunoreactive for glutathione S-transferase placental form (GST-P) and the numbers of proliferating and apoptotic cells. Co-treatment with EMIQ and ALA suppressed these increases. TAA-induced formation of p16(Ink4a-) foci in concordance with GST-P(+) foci was not suppressed by co-treatment with EMIQ or ALA. TAA-promotion increased cellular distributions of cell proliferation marker Ki-67, G2/M and spindle checkpoint proteins (phosphorylated checkpoint kinase 1 and Mad2), the DNA damage-related protein phosphorylated histone H2AX, and G2-M phase-related proteins (topoisomerase IIα, phosphorylated histone H3 and Cdc2) within GST-P(+) foci, and co-treatment with EMIQ or ALA suppressed these increases. These results suggest that downregulation of p16(Ink4a) may allow selective proliferation of preneoplastic cells by TAA promotion. However, antioxidants did not counteract this gene control. Moreover, effective suppression of TAA-induced cellular population changes within preneoplastic lesions by antioxidants may reflect facilitation of cell cycling and accumulation of DNA damage causing the activation of cell cycle checkpoints, leading to G2 and M phase arrest at the early stages of hepatocarcinogenesis promoted by TAA.

Topics: Animals; Carcinogenesis; Carcinogens; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Immunohistochemistry; Liver Neoplasms, Experimental; Male; Oxidative Stress; Precancerous Conditions; Rats; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide

The search for cancer specific nuclear proteins, stimulated by the supposition that transition from the normal to the neoplastic state resulting from disturbances in the control mechanisms of gene expression, indicated that non-histone protein of MW 48 kD is much more abundant in animal tumour cells than in normal liver (Krajewska et al., 1990). A non-histone component of MW 48 kD was assessed for changes during chemically induced carcinogenesis. Rats were treated with the hepatocarcinogen thioacetamide (TAA) and the expression of the polypeptide studied, in total nuclear protein and nonhistone protein fractions, was tested by Western blot technique in the presence of antibodies developed against a component of MW 48 kD from Kirkman-Robbins hepatoma. It was demonstrated that TAA-induced hepatocarcinogenesis was accompanied by the expression of non-histone protein of MW 48 kD at a significantly elevated level. A clear and distinct change in the expression of the component studied in the spleen of TAA-treated rats was also observed. These results support the suggestion that over-expression of non-histone protein of MW 48 kD could contribute to neoplastic transformation.

Topics: Animals; Cell Transformation, Neoplastic; Chromosomal Proteins, Non-Histone; Gene Expression Regulation, Neoplastic; Liver Neoplasms, Experimental; Male; Molecular Weight; Rats; Rats, Wistar; Thioacetamide

The results of an electron microscopic study of the changes in hepatocytes induced by chronic intoxication with thioacetamide are reported. During the poisoning aspecific toxic changes are intermingled with progressive, preneoplastic ones. The main cell subpopulations identified are: 1) large hepatocytes with smooth endoplasmic reticulum (SER) hypertrophy, with or without rough endoplasmic reticulum (RER) neoformation and glycogen storage, which is starvation resistant; 2) smaller hepatocytes, where RER hypertrophy and ribosome accumulation are the prominent features. Such a pattern persists for months. After the withdrawal of the drug most of the cell changes disappear. However, during this time a simplification of the liver structure and cell composition takes place, allowing a sequence of cell events which seem relevant for establishment of neoplastic progression. The SER-hypertrophied cell appears first and gives rise, via several intermediate stages, to the RER-hypertrophied one, which is believed to play a key role as the ultimate precursor of cancer cells.

Topics: Acetamides; Animals; Cell Transformation, Neoplastic; Endoplasmic Reticulum; Female; Hypertrophy; Liver; Liver Neoplasms; Microscopy, Electron; Necrosis; Precancerous Conditions; Rats; Thioacetamide; Time Factors

Cytoplasmic RNA from control and thioacetamide-intoxicated rat livers was compared with regard to components separated by polyacrylamide electrophoresis and for the proportion containing polyadenylic acid segments. A further comparison was made with the RNA's released from rat liver nuclei in vitro. Exposure in vivo to thioacetamide was associated with more cytoplasmic RNA's with migrations of 9 to 16 S and with both an acute and prolonged increase in the relative quantity of polyadenylic acid-containing polyribonucleotides. Incubation of control nuclei in vitro was associated with a leakage of approximately 1 to 2% of nuclear RNA with a major migration band at 9 S. Addition of adenosine 5'-triphosphate increased the transport 20-fold, and 9 and 16 S species were transported as well as a 4 S group. Following exposure to thioacetamide, an enhanced "leakiness" released 9 S RNA. Addition of adenosine 5'-triphosphate doubled the quantity of released RNA, which consisted of 9 and 4 S species. Both leaked and adenosine 5'-triphosphate-transported RNA's contained polyadenylic acid segments in roughly 20% of the macromolecules. These studies extend the observation of less stringent control of RNA release and transport in carcinogen exposure and suggest the potential usefulness of the in vitro release system for assaying cellular regulatory phenomena.

Topics: Acetamides; Adenosine Triphosphate; Animals; Carcinogens; Cell Transformation, Neoplastic; Cytoplasm; Electrophoresis, Polyacrylamide Gel; In Vitro Techniques; Liver; Liver Neoplasms; Male; Poly A; Rats; RNA, Neoplasm; Thioacetamide

Topics: Amines; Animals; Anthracenes; Azo Compounds; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Drug Evaluation, Preclinical; Embryo, Mammalian; Leukemia Virus, Murine; Mice; Mice, Inbred AKR; Naphthalenes; Neoplasm Transplantation; Nitrosamines; p-Dimethylaminoazobenzene; Quinolines; Thioacetamide; Transplantation, Homologous; Urethane